The present invention describes a new method for the production of certain carbohydrate containing compounds related to glycoconjugates; namely, lactosamine derivatives and substances derived therefrom. In a further aspect the present invention relates to products produced by the above method as well as uses of the resulting products.
Glycoconjugates contain saccharide chains with from one up to twenty monosaccharide units and in which certain sequences have been shown to have biological activity, for example in the binding of different cells, pathogens, toxins, as well as antibodies or other proteins to cell surfaces, in cancer metastasis, in inflammatory processes, for instance selectin-carbohydrate interactions in the binding of white blood cells to the blood vessel wall, as a modifier of the biological activity and stability of glycoproteins, as immunogenic substances, which have potential in the vaccination against different diseases (See for instance Annual Review of Biochemistry, vol. 58 (1989), pages 309-350, and Current Opinion in Structural Biology, for example review articles in vol. 3 (1993) and references therein).
Structures containing the sequence Galxcex21-4GlcNAc, called N-acetyl-lactosamine below, are especially of importance and are found for instance in glycoconjugate oligosaccharides of the lactosamine type. The structure is found in blood group structures, for instance Lewis-x (e.g. Galxcex21-4(Fucxcex11-3)GlcNAc), sialylated Lewis-x and 3xe2x80x2-sulfated Lewis-x, and is of importance in e.g. selectin-carbohydrate interactions (as reviewed by J. B. Lowe, in Molecular Glycobiology, pages 163-205, Fukuda and Hindsgaul, Eds., IRL Press at Oxford University Press, Oxford, 1994; see also Curr. Opin. Struct. Biol. vol. 3 (1993)).
It is of interest to be able to produce derivatives of lactosamine in large quantities for biological/clinical studies/tests, for example for inhibition of the selectin-carbohydrate interaction in vivo to inhibit/modify cell-mediated inflammatory processes (for instance in acute septic shock, ARDS, reperfusion injuries, rheumatoid arthritis, virus-induced pneumonia, psoriasis and the like).
Chemical methods known heretofore to produce N-acetyl-lactosamine and derivatives thereof have demanded multi-step synthesis and are often expensive and labor intensive. Enzymatic methods used before the present method were mainly based on the use of galactosyltransferase (EC 2.4), a cofactor dependent enzyme which requires UDP-galactose as a glycosyl donor (e.g. Wong et al., J. Org. Chem. (1982), pages 5416-5418). This type of enzyme also has disadvantages as high acceptor selectivity and exhibits low efficiency with unnatural acceptors, for instance derivatives of glucosamine (e.g. as reviewed by Khan and Hindsgaul in Molecular Glycobiology, pages 206-229, Fukuda and Hindsgaul, Eds., IRL Press at Oxford University Press, Oxford, 1994). For a general review of enzymatic methods, see K. G. I. Nilsson, Trends in Biotechnology, 1988, p. 256-264 (the nomenclature used in that review article and in this application follow the same IUPAC-rules). Glycosidases have been used to produce N-acetyl-lactosamine and N-acetyl-allolactosamine from galactosides and N-acetyl-glucosamine (Sakai et al., J. Carbohyd. Chem, 11: 553-565, 1992).
Earlier methods with glycosidases (EC 3.2) for production of derivatives of N-acetyl-lactosamine gave generally low yields because of low or wrong regioselectivity. Thus, for example, xcex1-galactosidase from E. coli or from ox-testes give solely Galxcex21-6GlcNPht, (K. G. I. Nilsson, unpublished result; Pht symbolizes a phthalimido group which generally is used as a temporary protection group on the amino group of glucosamine) when lactose is used as the glycosyl donor and GlcNPht is used as the acceptor.
The present invention describes a method which with unexpectedly high specificity gives the xcex21-4 linkage in the synthesis of different lactosamine derivatives, using abundant donor substances such as lactose and other low cost galactosyl donor substances. In one embodiment of the invention, the method is carried out by using the yeast Bullera singularis as a catalyst (classified as Bullera singularis according to Yeasts, second edition by Barnett et al., Cambridge University Press, 1990).
In a second embodiment, the process of the invention is carried out by using enzymes (which belongs to the group of glycosidases, EC Group 3.2), preferably in a crude, partially isolated or isolated form, especially xcex2-galactosidase from Bullera singularis but also other xcex2-galactosidase e.g. recombinant, of the same structure or of a similar structure (e.g., containing similar active site structure) as the one from Bullera singularis. 
According to the more detailed aspects of the present invention, the process for producing lactosamine derivatives can be carried out as an equilibrium (reversed hydrolysis) reaction or preferably as a kinetic (transglycosylation) reaction. As is known in the art, the principles of an equilibrium reaction and a kinetic reaction are well understood (e.g. see K. G. I. Nilsson, Trends in Biotechnol. (1988), pages 256-264).
In the case where the reaction is carried out as a transglycosylation reaction, the glycosyl donor is a glycoside, e.g. of D-galactose (Gal) modified in the C-1 position (anomeric position) but it can also be an oligosaccharide, such as lactose (Galxcex21-4Glc or aglycoside thereof, e.g.:
Galxcex2OR+GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3xe2x86x92Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3+ROH
R can be a glycosidically linked organic group, for example sugar (e.g. CnH2nOn or CnH2nxe2x88x922Onxe2x88x921 such as glucose), lower alkyl group (e.g. -Me, -Et) or an aromatic group (e.g. phenyl (-Ph), umberriferyl or m-, o-, or p-nitrophenyl group), preferably R is Glc (glucose) or nitrophenyl. Other glycosides (e.g. F-, N- or S-glycosides) may be selected.
It is known in the art that glycosidases allow some modification of the glycon part (i.e., the galactosyl part in the present invention) of the glycosyl donor. Therefore, in addition to Galxcex2OR, donors where the galactosyl part have been partially modified in a way still allowing the transglycosylation reaction to occur, resulting in the xcex21-4 linkage between the glycon part of the glycosyl donor and the glucosamine derivative, can be selected by the person skilled in the art for use with the method according to the present invention. Examples of such modifications of the glycon are modifications where at least one of the hydroxyl groups have inverted configuration (e.g. inversion in position 4 means that Gal is substituted for by Glc, that is Glcxcex2OR, i.e. a xcex2-glucoside, is used as donor), or where one of the hydroxyl groups of Gal has been modified or substituted for by an inorganic (e.g. -F, -H) or an organic group, e.g. a lower alkyl (e.g. methyl), allyl or an acetyl group. The selection of such a donor in the method according to the invention thus gives a xcex21-4 linked product in which the galactosyl part is correspondingly modified. Products of the type Rxe2x80x2-Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3 may thus be prepared where Rxe2x80x2-Gal relates to a modified glycon of the glycosyl donor. In the case of a transglycosylation reaction, the glycosyl donor in the scheme shown below, Rxe2x80x2-Gal-R, is a xcex2-glycoside:
Rxe2x80x2-Gal-R+GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3xe2x86x92Rxe2x80x2-Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3+RH
In the transglycosylation reaction,
Galxcex2OR+GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3xe2x86x92Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3+ROH
the reaction rate is higher than in the equilibrium reaction since the glycoside or disaccharide is more reactive than the non-activated sugar D-galactose used as donor in the equilibrium reaction. An enzyme of less purity, even a non-purified enzyme, can be used in the reaction since the enzymes are substrate/linkage specific and contaminating enzymes (e.g., xcex1-galactosidase) will not react with Galxcex2OR to give a xcex2-linked product. Thus, intact cells (e.g. yeast) can be used as well as partially purified enzyme or enzyme of higher purity.
Hydrolysis of Galxcex2OR will also occur to a certain extent depending on the reaction conditions. Lower or higher temperatures (e.g., room temperature or higher, e.g. 25xc2x0-65xc2x0 C.) can be selected, organic (co)solvents (acetone, acetonitrile, tetrahydrofurane) can be used, the pH typically is selected from the range 4 to 8, the substrate concentrations are typically 30 mM to several M concentration (e.g. 7 M) depending on the solubility of the substrates, stability of enzyme in the reaction mixture, and the particular goal of the reaction and the type of substrates.
The reaction will go through a maximum of product formation and has to be followed (e.g., preferably by HPLC) and terminated after an appropriate time by e.g. heat treatment at e.g. 80xc2x0-100xc2x0 C. for e.g. three minutes. Generally, donor consumption is tracked and the reaction terminated after a suitable time, which depends on the conditions, and often at xe2x89xa740% consumption of the donor. The reaction can be carried out for a few minutes to several hours depending on the growth of yeast cells (if fermentation conditions are used), the amount of enzyme, temperature, pH, concentration of substrates, and other factors.
The reaction can be monitored by means of TLC, HPLC or by spectrophotometric measurement of liberated aglycon (e.g. nitrophenol, 400 nm). Charring of TLC plates with sulfuric acid may be used for detection of sugars. When a desired yield of the product has been obtained, the reaction is terminated by denaturation of the enzyme by for example heat treatment. Heating to 85xc2x0 C. or above for 3-5 min (eventually followed by addition of ethanol to a concentration of about 80%) is usually sufficient. If immobilized enzyme is used, the reaction may be terminated by centrifugation or filtration.
In each type of reaction depicted above, a D-glucopyranosamino derivative is used as acceptor (GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3) where Rxe2x80x3 and Rxe2x80x2xe2x80x3 are defined below.
In the case of N-acetyl-glucosamine (Rxe2x80x3=xe2x80x94HAc group in the 2 position, i.e. in the N-position of 2-glucosamine; the 2-position in glucosamine contain a xe2x80x94NHAc), Rxe2x80x2xe2x80x3 represents an aglycon other than the anomeric hydroxyl group (i.e. Rxe2x80x2xe2x80x3 is not OH). If Rxe2x80x3 is not a xe2x80x94HAc group then Rxe2x80x2xe2x80x3 can be OH but can also represent a modification of the anomeric hydroxyl group as in the case where Rxe2x80x3 is xe2x80x94HAc.
The method according to the invention can be used to produce Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3 in high purity (no other linkages observed by 400 MHZ NMR), a product which after isolation can be used for biological/therapeutical purposes or for further synthesis according to the invention. The method can also be used to produce Galxcex21-4Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3. Thus, with the same enzyme and in a further reaction one will obtain Galxcex21-4Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3 as shown by the following equation:
Galxcex2OR+Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3xe2x86x92Galxcex21-4Galxcex21-4GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3
GlcNRxe2x80x3xe2x80x94Rxe2x80x2xe2x80x3, (the derivative of glucosamine which is used as acceptor), is of the general structure shown in the example below: 
where NRxe2x80x3 may be selected from compounds containing an inorganic group (e.g. N3, NHSO3H) and/or an organic group bound to the 2 position of glucosamine, such as (a) N-phthalimido; (b) an organic carbonyl group NHxe2x80x94C(O)xe2x80x94R where R is a hydrogen or a compound containing an organic group, e.g. aliphatics such as alkyl (e.g. methyl, ethyl, propyl), alkoxy (e.g. methoxy, ethoxy), allyloxy, amino acid or polypeptidyl is residue, and/or aromatics such as phenyl, benzyl or phenyloxy, preferred examples include N-chloromethoxyacetyl, N-phenoxyacetyl, NHBoc (Boc=t-butyloxycarbonyl), NHAc and NHC(O) (CH2)nCH3 (n is an integer equal to or greater than 1); (c) NHR where R is a compound containing an aliphatic and/or J aromatic group as described above, for example lower alkyl, preferred examples include NH(CH2)nCH3 (n is an integer equal to or greater than 1); or (d) NRRxe2x80x2 where R and Rxe2x80x2 are independently selected from compounds containing an aliphatic and/or aromatic group as described above; preferably NRxe2x80x3 is azido, 2-N-acetyl-, or 2-N-phthalimido;
and where Rxe2x80x2xe2x80x3 is selected from a glycosidically bound inorganic compound, e.g. fluoro or is selected from an O-, C-, N- or S-glycosidically bound compound containing an aliphatic and/or aromatic group, for example lower alkoxy (e.g. methyloxy (xe2x80x94OMe), ethyloxy (xe2x80x94OEt)), lower thioalkyl (e.g. xcex2-linked thioethyl (xe2x80x94SEt)), thioaromatic (e.g. thiophenyl-), xe2x80x94OEtBr, nitrophenoxy, amino acid, peptide, or derivative thereof, or another organic group of interest for the use of the product, or Rxe2x80x2xe2x80x3 can be xe2x80x94OH if NRxe2x80x3 is not NHAc but Rxe2x80x2xe2x80x3 is not xe2x80x94OH if NRxe2x80x3 is NHAc.
The Galxcex21-4GlcN (lactosamine) containing product obtained with Galxcex2OR as donor in the method according to the present invention is of the general structure shown in the example below: 
Such conjugates where lactosamine, or higher oligosaccharides containing the lactosamine structure, is N- or O-glycosidically bound to amino acids or peptide sequences, via the glucosamine residue in lactosamine and derivatives thereof as described above, are of interest to produce synthetically for fundamental studies and for synthesis of biologically/medically active fragments of glycoproteins, for instance to be used as vaccine or therapeutics. It is also important to be able to synthesize oligosaccharide analogues/derivatives of the structures above and according to the present invention to modify or improve the biological activity of the conjugate.
The synthetic procedure according to the invention can be carried out under highly diverse conditions in regards to, for example, pH, type of buffer, temperature and concentration of the reactants. Various cosolvents (N,N-dimethyl formamide, acetonitrile, dimethyl sulfoxide, dioxane, pyridine, methanol, ethanol, ethylene glycol, etc.) may be used and in varying concentrations together with water. In general, hydrophobic acceptor substances are more easily dissolved by the use of organic cosolvents or increased temperature. Moreover, the reactions may be carried out in two-phase systems, e.g. water-organic solvent or in two-phase systems of water-water polymer. The use of acceptors modified with organic groups facilitates recovery of the product in the organic phase.
The reaction conditions are not critical but are selected primarily on the basis of the properties of the reactants employed in the synthesis concerned, and also on the basis of practicality. For example, it may be mentioned that it is usually convenient to use a reaction temperature in the range of 25-75xc2x0 C. and, in the case of water-rich medium, the pH is usually in the range 4-8.
The reaction temperature may also be varied to influence product yield and the activity and stability of the enzyme and does not restrict the scope of the invention. The temperatures most frequently used lie in the range 4-75xc2x0 C., but lower temperatures and temperatures below 0xc2x0 C. can be used which can be facilitated if organic cosolvent is used. An advantage with high temperatures is, for example, that high substrate concentrations may be used, which reduces the water activity and thus increases the yield of product. Another advantage is that the activity of the enzyme increases, which means shorter reaction times at increased temperatures. The upper temperature limit is determined by the thermostability of the enzyme and substrate in the specific reaction medium. In some reactions the thermostability of the enzyme is increased by the use of high sugar substrate concentration. High concentration of substrate e.g. lactose ( greater than 15% w/w) can be achieved by dissolving in hot buffered water followed by cooling to the desired reaction temperature.
The concentration of the acceptor is a parameter which can be used to influence the yield of the reactions according to the invention. High concentrations are usually preferable in both equilibrium and transglycosylation reactions to minimize hydrolytic side-reactions, which usually means that depending on the solubility of the acceptor, ca. 0.05-7 M concentration of acceptor is used. In general, high concentrations of substrates are obtained by heating the reaction mixture to near the boiling point for a few minutes, allowing the solution to cool to the reaction temperature (usually 4-75xc2x0 C., depending on the temperature for optimum yield and thermostability of the enzyme/substrate) followed by addition of the enzyme. Cosolvents can be used to increase the solubility of substrates with hydrophobic groups.
The concentration of glycosyl donor in the reaction mixture is selected with regard to the lactosamine derivative to be synthesized and also with regard to the properties of the enzyme and therefore do not restrict the use of the invention. In some cases, addition of the donor in small portions may be advantageous in order to minimize the risk that the donor also acts as an acceptor (unless this is desired). Lactose is generally used as the donor since it is a cheap substrate. The weight ratio of donor to acceptor is preferably xe2x89xa71:1 though the acceptor can be in excess.
The enzyme may be used in situ or after partial or complete purification from its natural environment. The enzyme may be isolated before use by e.g. homogenization, precipitation and/or chromatography (e.g. based on ion-exchange, affinity, size). The enzyme may be present in e.g. soluble, immobilized, cross-linked, crystalline form or be enclosed within micelles. Generally, the glycosidase can be used in vivo or in vitro in a more or less purified form and in different cell types (as cloned into a suitable cell type). The enzyme may be produced with recombinant techniques. Then, if desired, one or more of the amino acids in the amino acid sequence of the enzyme may be changed in order to optimize the properties of the enzyme, e.g. thermostability, catalytic efficiency and/or stability in organic solvents. Variants of the glycosidase produced with recombinant technology which have at least 70% homology with the peptide chain of the natural variant are, together with the naturally occurring glycosidase, also useful according to the invention.
The synthetic reaction can be carried out with enzyme in vivo, that is under fermentation conditions with intact yeast cells and with lactose and acceptor in concentrations, for example, in the range 0.5 to 25% weight/volume. An excess of lactose is useful in some cases to improve the galactosylation of the acceptor and/or to prepare trisaccharides of the type mentioned above. This and other fermentation conditions with the necessary nutritional media/salts are easily determined by a person skilled in the art and does not limit the scope of the invention.
As glycosyl donor, lactose may be used or a xcex2-glycoside of galactose such as an alkyl or aromatic glycoside (e.g. nitrophenyl xcex2-galactoside).
Isolation of the product may be carried out in one or more steps involving one or more of the following procedures: extraction, chromatography (common solid supports that can be applied are e.g. Sephadex(copyright), silica, reversed-phase silica, charcoal, charcoal-celite), precipitation.
Depending on if intact yeast cells (fermentation conditions) or if crude, partially isolated or isolated enzyme are used and also with regard to the solubility and stability of substrates, the reaction may be carried out at different conditions and preferably under conditions most suitable for the particular reaction. Such conditions are chosen by the person skilled in the art and do not limit the scope of the invention. Conventional pH (e.g. 4-8 obtained by e.g. acetate or phosphate buffer) and at low temperature, room temperature or at increased temperatures (e.g. in the range 0-50xc2x0 C.) may be use if a crude, partially isolated, or isolated enzyme preparation is employed.
The reaction can be carried out in the presence of inert organic cosolvents in order to increase the solubility of the acceptor (e.g. hydrophobic acceptor) or to avoid hydrolysis reactions. If organic cosolvents (e.g. acetone, acetonitrile, tetrahydrofurane) are used together with buffered water as solvent for the reactions, lower temperatures than 0xc2x0 C. (e.g. xe2x88x9230xc2x0 C.) may be chosen in certain cases. The concentration of substrates are then usually in the range of 30 mM-7 M.
The enzyme may be used in soluble form or may be immobilized by e.g. adsorption, encapsulation, chelation, precipitation or covalent binding to a solid support, such as a polymeric substance, or a derivative thereof which is insoluble in protic or aprotic solvents (Methods in Enzymology, vol. 135, Academic Press). The form selected is not critical to the invention. If the enzyme is used in soluble form, it may if desired first have been chemically modified in a suitable manner in order to e.g. increase the thermostability or the stability in organic cosolvents. Enzyme immobilized to an insoluble polymer comprising, for example, agarose, cellulose, hydroxyethyl acrylate, glass, silica, polyacrylic amide, polyacrlyate-based plastics, etc., is readily separated from the product mixture, and the enzyme may thus be reused.
Examples of immobilization are adsorption or covalent binding of the enzyme to a suitable solid phase such as glass, celite, silica, polysaccharides (e.g. cellulose, agarose), or plastics (e.g. polystyrene), activated with a suitable reactive group for covalent binding of the enzyme as is known in the art (see e.g. Methods in Enzymology, volumes 44, 104 and 135).
If intact yeast cells are used, the reaction conditions are chosen by the person skilled in the art and do not limit the scope of the invention. Preferable conditions are normally pH 4-7, 20-35xc2x0 C. in buffered water containing nutrients for the yeast cells as exemplified in the non-limiting examples below.
Microorganisms which produce enzymes with the same structure or of a similar structure (e.g., containing similar three dimensional tertiary structure and active site structure) as the one from Bullera singularis can also be used.
If high concentrations of lactose are used, a considerable amount of glucose will be formed when a crude, partially purified or isolated enzyme is used (under fermentation conditions with intact yeast cells the yeast will consume a large amount of the formed glucose). The formed glucose will compete with the acceptor (and with water) for the galactosyl-enzyme intermediate, thus inhibiting the synthesis of product. A second enzyme which specifically removes glucose may thus be used during the reaction according to the invention, such as an isomerase (e.g. glucose isomerase) or an oxidase. Also, the product may be removed by the use of another specific enzyme, transferase, sulfatase which specifically converts the product to another desired product, thereby minimizing secondary hydrolysis of product and/or avoiding the need for isolation of the lactosamine product prior to its use in further synthesis.
The products can be used for further enzymatic synthesis with glycosidases or glycosyltransferases. For example, xcex1-sialyltransferase can be used to catalyze the formation of sialylated Gal-GlcNAc derivatives and xcex1-fucosyl transferase can be used to form oligosaccharide derivatives of the type Gal-(Fuc)GlcNAc-R, which then can eventually be sulphated, sialylated and/or be used for further chemical synthesis, etc.
The products obtained with the method according to the invention may be used directly for biological applications or may be used for further synthesis to obtain various lactosamine group containing products employing enzymatic and/or chemical methods (see e.g. Example 7 below) of interest for e.g. various clinical, diagnostic, downstream processing or for food supplement purposes. For references to chemical modification of glucosamine and examples of possible chemical conversions of modified lactosamines see e.g. Binkley: Modern Carbohydrate Chemistry, Marcel Dekker, 1988 with references; Paulsen, Chem. Soc. Rev. 13, pages 15-45; Khan and Hindsgaul in Molecular Glycobiology, pages 206-229, Fukuda and Hindsgaul Editors, IRL Press, Oxford. For a reference to the use of thioethyl glycosides in the synthesis of various glycosides or for use as glycosyl donors in convergent block synthesis of tri-, tetra- and larger saccharides, see e.g. references cited in the Khan and Hindsgaul article.
The product obtained according to the invention may also be converted by enzymatic methods using e.g. lipases, sulfatases, glycosyltransferases and oxidases. In this way hydroxyl groups of the galactosyl or glucosaminyl moiety may be selectively modified with e.g. acyl groups, sulphate groups, saccharide groups and other organic groups respectively, thus further extending the utility of the method of the invention for preparation of different derivatives and higher saccharides containing the lactosamine group. Specific examples are the selection of a suitable lactosamine derivative prepared by the method according to the invention for reaction with e.g. a sialyltransferase or sulfatase to obtain e.g. an (xcex12-3) sialylated lactosamine derivative or a 3xe2x80x2-O-sulphated derivative containing the lactosamine group, respectively. For references to enzymatic modifications, see e.g. Khan and Hindsgaul above (glycosyltransferases) and Wang and Whitesides in Enzymes in Synthetic Organic Chemistry, Pergamon (1994), Elsevier Science LTd.; see also Enzyme Nomenclature, Academic Press (1984).
The aglycon of the lactosamine containing product obtained according to the invention may not only be used in glycosylation reactions (for formation of other glycosides or for synthesis of oligosaccharides containing the lactosamine sequence) but may also be used for covalent binding to another molecule such as a protein, bead or a solid support and the resulting product may then be used for various purposes. Thus, nitrophenyl glycosides are for example useful after reduction to aminophenyl glycoside for covalent binding to various proteins or solid supports, which then may be used in diagnostic reagents, in down stream processing for separation of various proteins and enzymes including glycosyltransferases with specificity of various proteins and enzymes including glycosyltransferases with specificity of acceptors containing the lactosamine sequence or for solid phase synthesis of oligosaccharides (see e.g. Wong and Whitesides above for references to solid phase synthesis of oligosaccharides).